Nicotinamide regeneration

Oxidation of P-nicotinamide adenine dinucleotide (NADH) to NAD+ has attracted much interest from the viewpoint of its role in biosensors reactions. It has been reported that several quinone derivatives and polymerized redox dyes, such as phenoxazine and phenothiazine derivatives, possess catalytic activities for the oxidation of NADH and have been used for dehydrogenase biosensors development [1, 2]. Flavins (contain in chemical structure isoalloxazine ring) are the prosthetic groups responsible for NAD+/NADH conversion in the active sites of some dehydrogenase enzymes. Upon the electropolymerization of flavin derivatives, the effective catalysts of NAD+/NADH regeneration, which mimic the NADH-dehydrogenase activity, would be synthesized [3]. 

For the majority of redox enzymes, nicotinamide adenine dinucleotide [NAD(H)j and its respective phosphate [NADP(H)] are required. These cofactors are prohibitively expensive if used in stoichiometric amounts. Since it is only the oxidation state of the cofactor that changes during the reaction, it may be regenerated in situ by using a second redox reaction to allow it to re-enter the reaction cycle. Usually in the heterotrophic organism-catalyzed reduction, formate, glucose, and simple alcohols such as ethanol and 2-propanol are used to transform the... 

Pahnore GTR, Bertschy H, Bergens SH, Whitesides GM. 1998. A methanol/dioxygen biofuel cell that uses NAD -dependent dehydrogenases as catalysts Application of an electro-enzymatic method to regenerate nicotinamide adenine dinucleotide at low overpotentials. J Electroanal Chem 443 155-161. 

Human CYPs are multicomponent enzyme systems, requiring at a minimum the CYP enzyme component and a reductase component to be functional. The reductase requires a reduced nicotinamide cofactor, typically NADPH, and this cofactor must be regenerated to provide a steady supply of reducing equivalents for the reductase. Regeneration is accomplished with a separate substrate and enzyme. Glucose-6-phosphate and glucose-6-phosphate dehydrogenase have been widely used for this purpose. The overall complexity of the reaction mixtures and their cost have been barriers to the widespread use of recombinant human CYPs for metabolite synthesis in the past. 

In the processes that require regeneration of cofactors such as nicotinamide adenine dinucleotide phosphate (NAD(P)H) and adenosine triphosphate (ATP), whole-cell biotransformations are more advantageous than enzymatic systems [12,15]. Whole cells also have a competitive edge over the isolated enzymes in complex conversions involving multiple enzymatic reactions [14]. 

Transition Metal-Catalyzed Regeneration of Nicotinamide Cofactors... 

A number of photochemically or photoelectrochemically activated transition-metal complexes have also been used, both for oxidation and reduction of the nicotinamide cofactors. Among these complexes is the aforementioned Cp Rh(bpy)-complex 9 [52, 53]. For details of these systems or other regeneration procedures using special dyes, the reader is referred to other reviews on coenzyme regeneration [17, 21-23]. 

To facilitate its application in organic synthesis, we developed a lyophilized cell powder of Sphingomonas sp. HXN-200 as a biohydroxylation catalyst. Hydro-xylation of A-benzyl-piperidine with such catalyst powder showed 85% of the activity of a similar hydroxylation with frozen/thawed cells, shown in Figure 15.6. The fact that rehydrated lyophilized cells are able to carry out such a reduced nicotinamide adenine dinucleotide (NADH)-dependent hydroxylation indicates that these cells are capable of retaining and regenerating NADH at rates equal to or exceeding the rate of hydroxylation. To our knowledge, this is the first example of the use of lyophilized cells for a cofactor-dependent hydroxylation. 

New supramolecular compounds mimicking biological cofactors have been proposed which have high redox potentials and, further, can be Hnked covalently onto supports in biotransformation systems [52]. Bioelectrochemical methods available for regeneration of nicotinamide- and flavin-dependent systems are comprehensively reviewed by Kohhnan et al. [53]. 

In the second approach the reducing equivalents are suppHed by a nicotinamide cofactor (NADH or NADPH) and for commercial viability it is necessary to regenerate the cofactor using a sacrificial reductant ]12]. This can be achieved in two ways substrate coupled or enzyme coupled (Scheme 6.2). Substrate-coupled regeneration involves the use of a second alcohol (e.g. isopropanol) that can be accommodated by the KRED in the oxidative mode. A problem with this approach is that it affords an equilibrium mixture of the two alcohols and two ketones. In order to obtain a high yield of the desired alcohol product a large excess of the sacrificial alcohol needs to be added and/or the ketone product (acetone) removed... 

Another approach to preparing enantiomerically pure carboxylic acids and related compounds is via enanhoselective reduction of conjugated double bonds using NAD(P)H-dependent enoate reductases (EREDs EC 1.3.1.X), members of the so-called Old Yellow Enzyme family [44]. EREDs are ubiquitous in nature and their catalytic mechanism is well documented [45]. They contain a catalytic flavin cofactor and a stoichiometric nicotinamide cofactor which must be regenerated (Scheme 6.23). 

Chenault, H.K. and Whitesides, G.M. (1987) Regeneration of nicotinamide cofactors for use in organic synthesis. Appl Biochem Biotechnol, 14, 147-97. 

Enoate reductase [153, 154], which occurs in strains of Clostridium or Proteus, and 2-oxo-acid reductase [155] from Proteus vulgaris or P. mirabilis catalyzes the stereospecific reduction of substrates performed directly by reduced methyl-viologen. No nicotinamide coenzyme is required. Methylviologen is regenerable electrochemically. Examples of the reduction of enoates, ketones, and 2-oxo acids are given in [155]. 

Generally, the nicotinamide coenzymes are not covalently bound to the enzyme. They are employed in enzyme assays and preparative applications by adding catalytical but optimized amounts, and they need to be recycled. For an economic process, an efficient regeneration method is a basic requirement. The necessary recycle number depends essentially on the value of the chiral product, generally the method should recycle the coenzyme 100-100,000 times ([42]). 

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